Project Summary/Abstract Every cell has a complement of distinct actin-based structures that play specific roles. These structures help cells maintain integrity and polarity, they drive endocytosis and cytokinesis, and in the cases of motile and muscle cells, they are a major component of the contractile machinery. Actin nucleators, proteins that stimulate formation of new filaments, are essential determinants of actin architecture. Understanding the design principles that distinguish nucleators, such as formins, provides insight into how they build distinct structures. Formins in the Formin HOmology Domain (Fhod)-family play important roles in development, immunity, muscle maintenance, and other processes. Mammals have two Fhod-family formins, Fhod1 and Fhod3. Fhod3 is enriched in muscle cells, including cardiomyocytes, where it is critical for sarcomere development and maintenance. Fhod1 is predominantly expressed in non-muscle cells but is also expressed in muscle cells. In cardiomyocytes, Fhod1 is associated with non-sarcomeric structures, including costameres and intercalated discs, structures important for cellular integrity and mechanotransduction. Therefore, the cardiomyocyte is an ideal cell type in which to study how two closely related formins build distinct structures. Further, both Fhod1 and Fhod3 are implicated in human cardiomyopathies. Polymorphisms in Fhod3 are thought to account for 1-2% of hypertrophic cardiomyopathy cases. In dilated cardiomyopathies, increased expression of Fhod1 and decreased levels of Fhod3 are detected, consistent with the idea that the two formins play distinct roles. Early studies concluded that Fhod-family formins are not actin nucleators, unlike all other formins studied to date. Models were proposed in which barbed end capping and/or filament bundling are important Fhod activities. However, we recently demonstrated that Drosophila Fhod and human Fhod1 are, in fact, potent nucleators. Interestingly, they permit barbed end elongation but do not accelerate the growth rate, in contrast to most formins. Processive elongation is a hallmark of formins and is considered a mechanism by which formins build longer actin filaments than can be made by other nucleators. Both Fhod1 and Fhod3 are associated with small, actin-dense structures, some of which contain actin filaments of specific length, such as stress fibers and sarcomeres. We propose that nucleators designed to create shorter actin filaments are necessary to build these structures. We also discovered that Fhod1 discriminates between actin isoforms, nucleating non-muscle actin but not muscle actin. Thus we have a new perspective from which to study this important class of formins. We will test the hypotheses that strong nucleation, weak elongation, and isoform specificity are all activities necessary for Fhod1 and Fhod3 to build distinct actin-based structures in muscle cells. To do so, we will combine biochemical analysis with functional tests in human cardiomyocytes derived from stem cells. These cells are a powerful model in which to study muscle development, function, and disease. Thus this work can help us understand how Fhods function and are associated with cardiomyopathies.